The proposed research aims to enhance the structure-based understanding of molecular mechanisms in cell signaling at the membrane, focusing on functionally important structural elements of specific membrane proteins and their interaction partners in signaling mechanisms of drugs of abuse. A primary target are the structures of protein segments (termed loops) connecting the transmembrane (TM) segments in TM proteins, in the context of specific mechanistic hypotheses about the role these loops have in signaling protein-protein interactions. The motivation is that loops have been shown to contribute to the regulation of function of these proteins, e.g., in ligand recognition, signal transduction, substrate transport, making this project a fundamental and essential part of the ongoing effort to understand signaling mechanisms at the molecular level from a true structural and dynamic perspective; it will add invaluable information and tools without which this effort cannot succeed. Reliable loop structure calculation will be integrated into rigorous modeling of G Protein Coupled Receptor (GPCR) and Neurotransmitter transporters (NTs), in the context of specific biological problems and informed by the relevant experimental data. The proposed studies apply 3D modeling and computational analysis of the dynamics and energetics of the structural elements, with experimental probing and validation of the computational inferences in experimental assays of function. The combined computational/experimental approach is a paradigm for structure-function studies of a wide range of molecular mechanisms of cell function. The Specific Aims probe first the loops for which a specific functional role has been suggested in the regulation and action of GPCRs and NTs, to understand their specific contributions to the functional mechanisms, and to the responses elicited by drugs of abuse: Specific Aim 1: To calculate the structures of the loops that complete the models of specific GPCRs, including Dopamine D2 and D4 receptors and 5HT receptors to reveal mechanisms of both direct and indirect involvement in ligand binding, oligomerization, and interactions with G proteins and arrestin. A broadening of the scope to a class C GPCR (CRF1) is also planned. Specific Aim 2: To establish a protocol for the prediction of structural properties of loops in the neurotransmitter transporter (NT) proteins and apply the protocol to study computationally the involvement of key loops in the modulation of function by transporters including DAT, which is the target of cocaine and amphetamine. Specific Aim 3: To validate experimentally the findings of the computational probing of loop structure, and interactions with specific proteins that modulate GPCR and NT activity, to reveal and probe the functional involvement of the loops, and to integrate the structural and dynamic models into the mechanism of the complete system. BRET, scintillation proximity assays, and a variety of 1D and 2D mutagenesis studies will be used to study loop structure and protein and/or ligand interaction of dopamine receptors and transporters, serotonin receptors, and CRF1 - a class C GPCR.